This project will develop Diagnostics for Biodefense against Lassa virus (LASV), Junin virus and several other arenaviruses that induce severe, often fatal, hemorrhagic diseases, and are classified as Biosafety Level 4 and NIAID Biodefense Category A agents. The goal of our proposed study is to develop and validate multi-agent diagnostic immunoassays for arenaviruses. Specific Aim 1 is to clone cell lines producing murine and rhesus macaque monoclonal antibodies (MAb) specific for LASV glycoproteins and nucleoprotein. LASV glycoproteins GPC, GP1 and GP2, and the nucleoprotein (NP) will be expressed in bacteria or eukaryotic cell lines, and the recombinant proteins will be used to immunize mice and rhesus macaques. Specific Aim 2 is to develop LASV antigen-capture and immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody-capture enzyme-linked immunosorbent assays (ELISA). These assays will be directly compared for sensitivity and specificity to current assays based on BSL4 grown virus, and optimized for the ability to detect various strains of LASV, including Josiah, Macenta, Z132 and LP. Specific Aim 3 is to characterize the humoral immune response to LASV virus proteins in adult rhesus macaques and validate the antigen-capture and antibody-capture ELISA for rhesus serological samples. Cross-reactive epitopes of arenavirus glycoproteins and nucleoprotein based on murine and rhesus MAb and human antibodies from LASV infected patients will be identified, including early IgM specific epitopes that appear to be the most important diagnostically. Specific Aim 4 is to broaden the specificity of the antigen-capture and antibody-capture ELISA to include both Old World arenaviruses and New World arenaviruses with potential for use as bioweapons. Specific Aim 5 is to validate the antigen-capture and antibody-capture ELISA using confirmed clinical samples and control samples field-collected from Guinea, Sierra Leone and South America. The potential use of arenaviruses as biological weapons directed against civilian or military targets necessitates development of effective diagnostics. The multi-agent diagnostic immunoassays for arenaviruses would be used to determine the attack agent following a deliberate release, and allow the virus used to be distinguished from other hemorrhagic fever viruses such as dengue virus or Ebola virus that may have similar case presentations. Development of rapid immunodiagnostic assays will also improve treatment of arenaviral diseases, facilitate studies to understand their prevalence and natural history, and lead to vaccines for preventing these major causes of morbidity and mortality. [unreadable] [unreadable]